A method for constructing and applying an α-synuclein preformed fibrous mouse model of intestinal Parkinson's disease.

Abstract

This invention belongs to the field of experimental animal model construction technology, specifically relating to a method for constructing and applying an α-synuclein preformed fiber-induced intestinal Parkinson's disease mouse model. The method includes the following steps: performing vagotomy on mice, followed by injecting α-synuclein preformed fibers into the duodenal muscle layer. This invention systematically validates the model from three dimensions: pathology, behavior, and microbiome, providing new evidence for the "gut microbiota-gut-brain axis" in the intestinal α-synuclein preformed fiber injection model and providing experimental evidence for the Braak hypothesis.

Description

Technical Field

[0001] This invention relates to a method for constructing and applying an α-synuclein preformed fibrous mouse model of intestinal Parkinson's disease, belonging to the field of experimental animal model construction technology. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide and a representative disease characterized by the accumulation of α-synuclein (α-syn) in the brain. Pathological features of PD include significant loss of dopaminergic neurons in the substantia nigra, leading to dopamine deficiency in the basal ganglia and resulting in motor disorders such as bradykinesia, rigidity, resting tremor, and gait disturbances. Clinically, PD patients often exhibit non-motor symptoms such as sleep disturbances, depression, and constipation before the onset of motor disorders. Furthermore, early-stage PD patients show changes in their gut microbiota, suggesting that the gut may play a crucial role in the early pathogenesis of PD. Therefore, constructing an animal model that can simulate the transmission of α-syn from the gut to the brain is of great significance for further exploring the origin and pathogenesis of PD.

[0003] Currently, animal models used for PD research mainly include neurotoxin models, genetic models, and α-synuclein inoculation models. In neurotoxin models, dopaminergic neurons are selectively damaged using toxins such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or rotenone. 6-OHDA is primarily used for rat PD modeling, involving large-scale destruction of dopaminergic neurons in the substantia nigra and striatum. It is mainly used to study the motor and biochemical dysfunctions in PD, but it does not induce α-synuclein deposition. Furthermore, 6-OHDA cannot cross the blood-brain barrier and must be injected via stereotactic brain surgery. MPTP induces specific loss of substantia nigra and striatum neurons in many vertebrate species, from humans to mice, through oxidative stress, mitochondrial apoptosis, and inflammation. It also does not induce α-synuclein deposition, and MPTP has shown the ability to induce PD in humans. However, it is easily absorbed through the skin, requiring high levels of safety precautions for researchers. Rotenone is a classic inhibitor of mitochondrial complex I, which can induce the formation of α-synuclein aggregates and the death of dopaminergic neurons. The advantage of these neurotoxin models is that they can rapidly induce damage to substantia nigra striatal neurons, but most of them cannot simulate the core pathological feature of α-syn aggregation, and the damage to dopaminergic neurons is often too acute, which is inconsistent with the chronic progression of PD.

[0004] In genetic models, the autosomal dominant genes are SNCA and LRRK2, while the autosomal recessive genes are mainly PRKN, PINK1, and DJ-1. In the SNCA gene model, the A53T transgenic mouse is widely used. This mouse overexpresses human α-syn, exhibiting significant α-syn accumulation in neuronal cell bodies. Motor impairment begins to appear at 8 months of age, eventually leading to neuronal dysfunction and death. It displays α-syn pathology observed in humans, including α-syn accumulation, α-syn phosphorylation and ubiquitination, and progressive age-dependent neurodegeneration. It shows a high degree of similarity to human PD pathology and is one of the key models for PD research.

[0005] In the α-synuclein inoculation model, recombinant α-synuclein monomeric proteins are treated under specific conditions to generate aggregated α-synuclein preformed fibrils (PFFs), whose structure is similar to Lewy bodies (LBs). These short fibrils, generated by ultrasound, are highly pathogenic. Stereoscopic injection of PFFs into the brain successfully induces the accumulation of α-synuclein pathology and the loss of dopaminergic neurons. However, this model cannot focus on gastrointestinal α-synuclein pathology, limiting research on enterogenic PD.

[0006] In recent years, to investigate the hypothesis that PD originates from the gut, researchers have applied the α-syn PFF inoculation model to gastrointestinal injection, successfully establishing an enterogenic α-syn transmission model and providing an effective model tool for studying the Braak hypothesis. However, the α-syn PFF intestinal muscle layer injection model is affected by many factors, including injection site, injection dose, species, age, and genetic background. Current gastrointestinal α-syn PFF injection models are still in the early validation stage and have several unresolved key issues. Results reported in different studies show significant contradictions: for example, injection of α-syn PFF into the pylorus and duodenal muscle layer of mice can lead to the accumulation of phosphorylated α-syn and motor impairment in the brain within several months; however, injection of the same substance into the colon of rats and rhesus monkeys did not show any clear pathological changes in the brain even after 12 months. Furthermore, studies have demonstrated that age is a key factor affecting the efficiency of α-syn transmission to the brain after intestinal seeding, and aging may increase susceptibility to α-syn misfolding. More importantly, most existing models are established in wild-type animal settings, and genetic susceptibility is one of the most important risk factors for PD, especially for individuals carrying SNCA gene mutations such as A53T. To date, it remains unclear how genetic background affects the efficiency of gut-brain transmission of α-syndrome, and questions regarding whether α-syndrome precisely spreads from the gut to the brain via the vagus nerve and whether the gut microbiota plays a regulatory role in this process have not been clearly verified.

[0007] In summary, existing gastrointestinal α-syn PFF injection models have significant limitations in terms of experimental stability and the influence of age and genetic background on pathological transmission. Therefore, they remain unreliable tools for studying the pathogenesis of enterogenic PD in genetically susceptible individuals such as those with A53T. Consequently, there is a need to construct an animal model that can stably simulate the transmission of α-syn from the gut to the brain and is suitable for genetically susceptible individuals, in order to provide a more effective experimental tool for studying the early pathogenesis of PD. Summary of the Invention

[0008] In view of the above-mentioned technical problems in the prior art, the present invention provides a method for constructing and applying an α-synuclein preformed fibrous mouse model of intestinal Parkinson's disease.

[0009] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein preformed fibers, comprising the following steps: performing vagotomy on mice, and then injecting α-synuclein preformed fibers into the duodenal muscle layer of the mice.

[0010] Furthermore, the mice are C57BL / 6J mice or A53T α-synuclein transgenic mice.

[0011] Furthermore, the mice in question are A53T α-synuclein transgenic mice.

[0012] Furthermore, the injection is a multi-point injection, with a total injection dose of 25μg-40μg.

[0013] Furthermore, the multi-point injection involves injecting at 3-7 sites, preferably at 5 sites.

[0014] Furthermore, the preparation of the α-synuclein pre-fibrillated injection solution includes: dissolving recombinant human monomeric α-synuclein in PBS, shaking and incubating for 6-8 days to obtain α-synuclein pre-fibrillated stock solution, then diluting the α-synuclein pre-fibrillated stock solution with PBS to the required concentration, and then performing ultrasonic treatment.

[0015] Furthermore, the concentration of the α-synuclein pre-fibrillation solution is 4 μg / μL-6 μg / μL, preferably 5 μg / μL, and it is diluted with PBS to a concentration of 2 μg / μL-3 μg / μL, preferably 2.5 μg / μL.

[0016] Furthermore, the vagotomy is a bilateral gastric vagotomy.

[0017] Furthermore, the transection site of the vagotomy is located at the junction of the esophagus and stomach.

[0018] Secondly, the present invention provides the application of the above-mentioned method for constructing an α-synuclein preformed fibrous induced intestinal Parkinson's disease mouse model, and uses the constructed intestinal Parkinson's disease mouse model to study the intestinal-brain transmission mechanism of Parkinson's disease, the correlation between intestinal flora and Parkinson's disease, or to screen anti-Parkinson's disease drugs.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The model constructed by the method of the present invention demonstrates that injecting α-synuclein preformed fibers into the duodenal muscle layer of C57 mice can induce phosphorylated α-synuclein pathology to spread upward along the vagus nerve to the central nervous system and cause motor disorders, thus confirming that the vagus nerve is a necessary pathway for pathological transmission.

[0020] This invention is the first to discover that α-synuclein preformed fibers injected into the duodenal muscle layer cause Parkinson's disease-like intestinal flora dysbiosis, and demonstrates that vagotomy has a selective regulatory effect on the flora.

[0021] The construction method of this invention was applied to A53T transgenic mice, demonstrating that the A53T genetic background can exacerbate the pathological spread of phosphorylated α-synuclein from the intestine to the brain via the vagus nerve, leading to more severe Parkinson's disease-like symptoms.

[0022] This invention systematically validates the model from three dimensions: pathology, behavior, and microbiome, providing new evidence for the "gut microbiota-gut-brain axis" of the gut-derived α-synuclein preformed fibrous injection model and providing experimental evidence for the Braak hypothesis. Attached Figure Description

[0023] Figure 1 A schematic diagram of a procedure involving duodenal muscle layer injection and bilateral gastric vagotomy. Figure 2 Immunohistochemical staining of phosphorylated α-synuclein in the duodenum 4 months after injecting α-synuclein into C57 mice to preform fibrosis; Figure 3 Image showing the amount of p-α-syn deposition in C57 mice 4 months after injection of α-synuclein pre-fibrils; Figure 4 HE staining of the duodenum of C57 mice 4 months after injection of α-synuclein to preform fibrous cells; Figure 5 Pathological score of duodenal tissue 4 months after C57 mice were injected with α-synuclein to preform fibrous tissue; Figure 6Tensile test results of C57 mice 3 and 4 months after injection of α-synuclein to pre-form fibers; Figure 7 Results of rotor drop latency in C57 mice 3 and 4 months after injection of α-synuclein to pre-form fibers; Figure 8 Forelimb stride data of C57 mice 4 months after injection of α-synuclein pre-fibers; Figure 9 Data on hind limb stride in C57 mice 4 months after injection of α-synuclein pre-fibers; Figure 10 Forelimb gait data of C57 mice 4 months after injection of α-synuclein pre-fibers; Figure 11 Data on hind limb gait speed in C57 mice 4 months after injection of α-synuclein pre-fibers; Figure 12 Timing diagram of mouse gait in C57 mice 4 months after injection of α-synuclein pre-fibers; Figure 13 Western blot was used to detect the expression level of p-α-syn in the striatum of C57 mice 4 months after injection of α-synuclein prefibrils. Figure 14 The expression level of p-α-syn in the striatum of the brain after injecting α-synuclein pre-fibers into C57 mice; Figure 15 TH immunohistochemical staining image of C57 mice after injection of α-synuclein to pre-form fibrous fibers; Figure 16 Number of TH-positive cells in the substantia nigra region of C57 mice after injection of α-synuclein pre-fibers; Figure 17 Phylogenetic composition of feces in C57 mice 4 months after injection of α-synuclein prefibrils; Figure 18 Species-level microbial composition of feces in C57 mice 4 months after injection of α-synuclein prefibrils; Figure 19 Analysis of LEfSe levels in PBS and PFF groups in Example 1; Figure 20 Analysis of LEfSe levels in the PFF and TV+PFF groups in Example 1; Figure 21 KEGG pathway enrichment analysis in the PBS and PFF groups of Example 1; Figure 22 KEGG pathway enrichment analysis for the PFF and TV+PFF groups in Example 1; Figure 23 Enrichment analysis of the MetaCyc metabolic pathway in the PBS and PFF groups in Example 1; Figure 24 MetaCyc metabolic pathway enrichment analysis for the PFF and TV+PFF groups in Example 1; Figure 25 Immunohistochemical staining of phosphorylated α-synuclein in the duodenum 4 months after injection of α-synuclein pre-fibrils into A53T transgenic mice; Figure 26 Image showing the amount of p-α-syn deposition in A53T transgenic mice 4 months after injection of α-synuclein pre-fibrils; Figure 27 HE staining of the duodenum of A53T transgenic mice 4 months after injection of α-synuclein to preform fibrous cells; Figure 28 Pathological score of duodenal tissue 4 months after injection of α-synuclein pre-fibrils into A53T transgenic mice; Figure 29 The amount of p-α-synuclein aggregation in the brain of A53T transgenic mice 4 months after injection of α-synuclein pre-fibrils; Figure 30 Tensile test results of A53T transgenic mice 3 and 4 months after injection of α-synuclein to pre-form fibers; Figure 31 Representative immunofluorescence images of Iba-1 (green), GFAP (red), and DAPI (blue) in the midbrain of A53T transgenic mice after injection of α-synuclein pre-fibrils; Figure 32 Number of TH-positive cells in the substantia nigra region of the brain after injection of α-synuclein pre-fibers into A53T transgenic mice. Detailed Implementation

[0024] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0025] Example 1 (1) Preparation of α-synuclein preformed filaments (α-synuclein PFF) Recombinant human monomeric α-synuclein (CF66, purchased from Suzhou Nearshore Protein Technology Co., Ltd.) was dissolved in PBS at a concentration of 5 μg / μL. The solution was placed in a 37°C constant temperature shaker and shaken at 1000 rpm for 7 days to prepare α-synuclein pre-fibrillation stock solution, which was then stored at -80°C.

[0026] Before use, thaw the α-synuclein pre-fibrillated protein solution at room temperature, dilute it with PBS to the required concentration (2.5 μg / μL), and perform sonication. The specific operation is as follows: perform 60 cycles at 20% power (sonication time 30 seconds, 0.5 seconds on, 0.5 seconds off).

[0027] (2) Injection of α-synuclein into the duodenal muscle layer of C57 mice to preform fibrous tissue Laboratory animals: Strain: C57BL / 6J mouse; Age in weeks: 8 weeks; Sex: Male; Weight: Approximately 24g; Rearing environment: The rearing environment is clean, with a temperature of 24±2℃, and a 12-hour light-dark cycle. Food and drinking water are plentiful.

[0028] See Figure 1 The surgical procedure is as follows: Mice were anesthetized by intraperitoneal injection of a combination of anesthetics (Shutai 50: 20.8 mg / kg; Xylazine hydrochloride: 2.7 mg / kg, diluted with physiological saline, administration volume 5 mL / kg).

[0029] PFF group mice were injected with α-synuclein preformed filaments. The procedure was performed under a stereomicroscope, using a 34g microsyringe to inject α-synuclein preformed filaments (injection concentration 2.5μg / μL) into the duodenal muscle layer of mice. 2.5μL was injected at each of the 5 sites, 0.5cm apart, for a total injection of 31.25μg.

[0030] Mice in the PBS group (blank control group) were injected with an equal volume of PBS.

[0031] After injection, the abdominal muscle layer and skin layer were sutured separately using 4-0 absorbable surgical sutures. Iodine solution was applied evenly after suturing, and the mice were placed back in their cages in a supine position to avoid pressure on the wound.

[0032] TV+PFF group: Mice underwent gastric vagotomy. Specifically, the esophagus connecting to the stomach was gently pried open with a glass needle, revealing the vagus nerve tightly attached to the esophagus. The bilateral vagus nerves were then grasped and severed with fine forceps. The severance site was located at the junction of the esophagus and stomach, slightly away from the stomach and towards the neck, approximately 0.5 cm above the stomach in the esophagus, to prevent incomplete vagus nerve severance. PFF was then injected immediately, with the same procedure and injection parameters as the PFF group. The suture was then performed after injection.

[0033] (3) Detection of intestinal inflammation after injection of α-synuclein into the duodenal muscle layer of C57 mice to preform fibers Four months after injection of α-synuclein pre-fibrils, p-α-synuclein pathology in the duodenum and brain of mice was examined. The PFF group showed significant p-α-synuclein deposition in the duodenal muscle layer, and phosphorylated α-synuclein expression was significantly increased compared to the PBS group (see [link to PFF group]). Figure 2 , Figure 3 HE staining results showed that, compared with the PBS group, the PFF group had disrupted small intestinal villus structure, significantly shortened villi, atrophied and loosely arranged crypts, obvious vacuolation at the villus tips, and a reduced number of goblet cells; a small number of inflammatory cells were observed infiltrating, indicating that the injection of pre-formed fibers into the duodenal muscle layer 4 months later led to the destruction of the normal small intestinal tissue structure. The elevated histopathological score indicated an intestinal inflammatory response (see [link to article]). Figure 4 , Figure 5 ).

[0034] (4) Motor behavior detection after injection of α-synuclein into the duodenal muscle layer of C57 mice to preform fibers Motor behavior tests were performed 3 and 4 months after the injection of α-synuclein pre-fibers.

[0035] No significant motor impairment was observed in the mice 3 months after injection.

[0036] See Figures 6-7 Four months after injection, mice began to exhibit motor dysfunction. Compared with the PBS group, the PFF group showed a significant decrease in pulling force and rotarod drop latency, indicating weakened motor ability, while the TV+PFF group showed a significant increase in pulling force and rotarod drop latency, indicating improved motor function.

[0037] See Figures 8-11 In the gait test, compared with the PBS group, the stride length of the forelimbs and hindlimbs of the mice in the PFF group was significantly reduced, and the forelimb gait speed showed a decreasing trend. In contrast, the stride length of the hindlimbs and the gait speed of the mice in the TV+PFF group were significantly increased. No significant difference was observed in the hindlimb gait speed among the three groups of mice.

[0038] The gait timing diagram also showed that, compared with the PBS group, the PFF group had a shorter stride and more irregular footprints, while the TV+PFF group had a significantly longer stride and more regular footprints. This indicates that vagotomy can effectively improve gait disorder in mice caused by injection of α-synuclein preformed fibers into the duodenal muscle layer (see [link to relevant documentation]). Figure 12 ).

[0039] (5) Pathological examination of the brain after injection of α-synuclein pre-fibers into the duodenal muscle layer of C57 mice To further investigate whether p-α-syn can spread from the duodenum to the brain, the expression level of p-α-syn in the striatum of mouse brains was detected by Western blotting. It was found that the expression level of p-α-syn in the striatum of the PFF group was significantly increased compared to the PBS group, while the expression level of p-α-syn in the TV+PFF group was significantly decreased (see [link to study]). Figure 13 , Figure 14 This indicates that pathological p-α-syndrome can spread from the intestines to the brain via the vagus nerve, and cutting the vagus nerve can block the enterobrain transmission of p-α-syndrome.

[0040] Immunohistochemical results of TH showed that the number of TH-positive cells in the substantia nigra was significantly reduced in the PFF group, while vagus nerve transection significantly reduced the loss of TH-positive cells and improved motor dysfunction in mice. Figure 15 , Figure 16 ).

[0041] (6) Analysis of gut microbiota after injection of α-synuclein into the duodenal muscle layer of C57 mice to preform fibers Four months after injecting α-synuclein pre-fibrils into the duodenal muscle layer of C57 mice, mouse feces were collected for metagenomic sequencing analysis.

[0042] The results show: At the phylum level: The PFF group exhibited dysbiosis characteristics highly consistent with those of PD patients, with significantly increased abundance of Pseudomonadota and Actinomycetota. Notably, the abundance of Verrucomicrobiota was decreased, contrary to the increased abundance of this phylum reported in some PD studies, suggesting possible species-specific differences or disease stage-dependent changes. Vagotomy had a reversal effect on the abundance changes of the above three phyla, but simultaneously exacerbated the enrichment of Bacteroidota and the reduction of Bacillota and Campylobacterota, suggesting that the regulatory effect of vagotomy on the microbiota is not a simple reversal, but a complex remodeling (see [link to relevant documentation]). Figure 17 ).

[0043] Level: The PFF group showed an increased abundance of compensatory bacteria such as Paramuribaculum intestinale, Muribaculumintestinale, and Muribaculum gordoncarteri.

[0044] Intestinal para-mouse bacillus colonization can partially mimic the metabolic regulation and barrier protection effects of Bupleurum polysaccharide, effectively improving the disease phenotype of mice with fatty liver disease associated with metabolic dysfunction, and synergistically enhancing the hepatoprotective effect of Bupleurum polysaccharide on mice with metabolic dysfunction-related fatty liver disease. Intestinal para-mouse bacillus inhibits the colonization of Salmonella typhimurium by converting succinate to propionate, maintaining intestinal barrier function and playing an important role in restoring colonization resistance. Gordoncatcher bacillus can metabolize to produce uric acid, reduce the infiltration of myeloid-derived suppressor cells, activate CD8+ T cells, and promote the transition from an immunosuppressive tumor microenvironment to an immune-active state.

[0045] Meanwhile, the abundance of probiotics such as Akkermansia muciniphila, Ligilactobacillus murinus, and Limosilactobacillus reuteri decreased in the PFF group.

[0046] Akkermansia myxophilus is a mucus-degrading symbiotic bacterium with unique physiological characteristics. By expressing specific functional proteins such as Amuc_1100, it exhibits strong anti-inflammatory and metabolic regulatory capabilities, directly participating in the regulation of mucosal homeostasis. Lactobacillus is an important lactic acid-producing flora in the gut, and has been confirmed in multiple studies to have immunomodulatory and anti-inflammatory effects. Lactobacillus murineis can alleviate croton-induced intestinal toxicity and works synergistically with processed croton oil to more effectively treat ulcerative colitis. Lactobacillus reuteri, a widely used probiotic, can maintain the intestinal barrier and immune homeostasis, regulating local and systemic immune responses under stable conditions and complex pathological conditions. A reduction in these probiotics may directly lead to a decline in intestinal immune regulation and impaired intestinal barrier function.

[0047] Vagotomy selectively, rather than comprehensively, modulates the gut microbiota. On one hand, it successfully reverses the enrichment of some pathogenic bacteria induced by α-synuclein prefibrillary fibrosis, such as *Paragonimus westermani* and *Rhizobium spp.*, and increases the abundance of beneficial bacteria such as *Akkermansia myxophilus*, *Lactobacillus murineis*, and *Lactobacillus johnsonii*. On the other hand, vagotomy exacerbates the enrichment of some commensal bacteria, such as *Duncas dubosii*, and leads to a further reduction in lactic acid-producing beneficial bacteria such as *Lactobacillus taiwanensis* (see [link to article]). Figure 18 ).

[0048] To compare the differences in gut microbiota composition among groups, LEfSe analysis was performed. (See [link to LEfSe analysis]) Figures 19-20The results showed that compared with the PBS group, the PFF group had a significant increase in the abundance of 29 bacterial species and a significant decrease in the abundance of 6 bacterial species. In contrast, the TV+PFF group had a significant increase in the abundance of 31 bacterial species and a significant decrease in the abundance of 127 bacterial species compared with the PFF group. The top 15 bacterial species were visualized, revealing an increase in *Bacteroides cellulosilyticus*, *Bacteroides intestinalis*, *Bacteroides zoogleoformans*, *Desulfovibrio fairfieldensis*, *Mucilaginibacter celer*, *Parabacteroides chongii*, and *Proteiniphilum propionicum*. The degree of change in gut microbiota composition caused by vagotomy even exceeded the effect of α-synuclein preformed fibers, suggesting that vagotomy does not simply reverse dysbiosis, but rather reshapes the gut microbiota structure, thus affecting gut microbiota composition. The regulation of the brain axis has produced complex effects.

[0049] To explore potential functional differences in gut microbiota among groups, statistical analysis of HUMAnN-predicted KEGG and MetaCyc was performed using STAMP software. KEGG results showed that, compared to the PBS group, the PFF group exhibited significant downregulation of core metabolic pathways such as ribosome synthesis, purine / pyrimidine metabolism, and glycolysis / gluconeogenesis, suggesting that α-syn PFF-induced gut pathology may inhibit the basal metabolic activity of microorganisms. Following vagotomy, these pathways were significantly upregulated, indicating that trunk vagotomy, to some extent, restored the energy metabolism and synthesis functions of microorganisms, and had a certain ameliorative effect on changes in the gut microbiota (see [link to relevant documentation]). Figures 21-22 ).

[0050] MetaCyc metabolic pathway enrichment analysis further revealed functional changes in amino acid and cell wall synthesis. In the PFF group, the biosynthetic pathways of L-isoleucine, L-lysine, dTDP-β-L-rhamnose, coenzyme A, and peptidoglycan were generally downregulated. The downregulation of branched-chain amino acid (BCAA) biosynthesis pathways is particularly noteworthy. The levels of BCAAs in the plasma of PD patients are significantly decreased, worsening with the severity of PD. Since BCAAs in the human body mainly originate from dietary nutrients, and the gut microbiota is a key factor in the metabolism of dietary components, changes in BCAA levels in the plasma of PD patients may be related to the gut microbiota. Furthermore, the downregulation of the dTDP-β-L-rhamnose and peptidoglycan biosynthesis pathways suggests that α-syn PFF may inhibit bacterial cell wall synthesis, affecting the colonization capacity of the bacterial community. After vagotomy, these pathways were partially upregulated, especially the dTDP-β-L-rhamnose and coenzyme A biosynthesis pathways, which showed the most significant recovery (see [link to relevant documentation]). Figures 23-24 ).

[0051] In summary, duodenal muscular α-syn PFF injection not only induced intestinal p-α-syn pathology but also triggered dual dysregulation of gut microbiota structure and function by affecting the gut microenvironment. Notably, the increased abundance of protective bacteria such as *Rhizobium oryzae* and *Goldencatella oryzae* in the PFF group may represent a compensatory protective response of the gut microbiota to p-α-syn pathology. However, this compensation is incomplete; KEGG and MetaCyc analyses showed that the overall metabolic function of the gut microbiota remained largely suppressed, and the significant reduction in probiotics weakened the gut's defense function. In contrast, vagotomy remodeled the gut microbiota structure, regulated its metabolic function, alleviated the α-syn PFF-induced intestinal metabolic disturbances, and exerted complex effects on the gut-brain axis. This indicates that the vagus nerve is not only a passive pathway for pathological transmission but can also actively regulate the structure and function of the gut microbiota, resulting in complex influences on the gut environment.

[0052] Example 2 Construction of A53T transgenic mouse model: The mouse model was constructed using homozygous A53T transgenic mice according to the method in Example 1.

[0053] Experiments have shown that adjusting the number of injection sites to 3-7, the total dose to 25-40 μg, and the concentration to 2-3 μg / μL all successfully induced the model, with results comparable to those in the specific examples. Those skilled in the art can optimize the selection within the above ranges according to actual needs (such as mouse strain and age).

[0054] Motor behavior tests were performed at 3 and 4 months post-injection, and the results showed: Similar to the results in C57 mice, four months after injection, homozygous A53T transgenic mice showed significant p-α-syn accumulation in the duodenal muscle layer, but the p-α-syn level in the TV+PFF group was significantly lower than that in the PFF group. Figures 25-26 ).

[0055] HE staining results showed that the PFF group had severely atrophied and flattened intestinal villi, with almost no intact villi visible. The number of goblet cells and crypts was significantly reduced, and the intestinal tissue structure was severely damaged, resulting in a significantly higher histopathological score. In contrast, the TV+PFF group had a more intact structure, with more tightly packed intestinal villi visible. The number of goblet cells and crypts was also increased. Although there was slight vacuolation at the villus tips, the histopathological score was significantly lower (see [link to article]). Figures 27-28 ).

[0056] Furthermore, even when injected with the same amount of preformed fibers as C57 mice and harvested at roughly the same time, homozygous A53T transgenic mice showed extensive p-α-syn accumulation in the substantia nigra (SNc region) and surrounding brain regions, while the p-α-syn level in the brain of the TV+PFF group was significantly lower than that of the PFF group (see [link to original text]). Figure 29 This may be because homozygous A53T transgenic mice themselves have an overexpression of endogenous α-syn, while exogenous injection of PFF induces more misfolding of endogenous α-synuclein. However, this effect is vagus nerve dependent, indicating that the vagus nerve is a necessary condition for the transmission of p-α-syn from the gut to the brain.

[0057] Four months after injection of pre-formed fibers into the duodenal muscle layer, homozygous A53T transgenic mice also showed decreased motor function, with the PFF group exhibiting a significantly lower grip strength compared to the PBS group. Vagotomy improved the motor impairment caused by α-synuclein pre-formed fibers (see [link to original text]). Figure 30 Consistent with this phenomenon, the number of TH-positive cells in the substantia nigra and ventral tegmentum was significantly reduced in the PFF group, while the TV+PFF group reversed the loss of TH-positive cells caused by preformed fibers. Figures 31-32 ).

[0058] The construction method of this invention successfully constructed an enterogenic PD model, providing clear evidence for the pathology of enterogenic PD. It verified the Braak hypothesis in terms of gut microbiota, behavior, and pathology, and filled the gap in the A53T genetic background combined with α-synPFF injection model, providing a new option for future PD mechanism research and drug development.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing an α-synuclein preformed fibril-induced enteric Parkinson's disease mouse model, characterized by, The procedure includes the following steps: performing a vagotomy on mice and then injecting α-synuclein pre-fibers into the duodenal muscle layer of the mice.

2. The method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein pre-fibril formation according to claim 1, characterized in that, The mice were C57BL / 6J mice or A53T α-synuclein transgenic mice.

3. The method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein pre-fibril formation according to claim 2, characterized in that, The mice in question are A53T α-synuclein transgenic mice.

4. The method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein pre-fibril formation according to claim 1, characterized in that, The injection is a multi-point injection, with a total injection dose of 25μg-40μg.

5. The method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein pre-fibril formation according to claim 4, characterized in that, The multi-point injection refers to injection at 3-7 sites.

6. The method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein pre-fibril formation according to claim 5, characterized in that, The preparation of the α-synuclein pre-fibrillated injection solution includes: dissolving recombinant human monomeric α-synuclein in PBS, shaking and incubating for 6-8 days to obtain α-synuclein pre-fibrillated stock solution, then diluting the α-synuclein pre-fibrillated stock solution with PBS to the required concentration, and then performing ultrasonic treatment.

7. The method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein pre-fibril formation according to claim 6, characterized in that, The concentration of the α-synuclein pre-fibrinogen solution was 4 μg / μL-6 μg / μL, and it was diluted with PBS to a concentration of 2 μg / μL-3 μg / μL.

8. The method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein pre-fibril formation according to claim 1, characterized in that, The vagotomy is a bilateral gastric vagotomy.

9. The method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein pre-fibril formation according to claim 8, characterized in that, The transection site for the vagotomy is located at the junction of the esophagus and stomach.

10. The application of the method for constructing a mouse model of intestinal Parkinson's disease induced by α-synuclein pre-fibril formation as described in any one of claims 1-9, characterized in that, The mouse model of intestinal Parkinson's disease constructed by them was used to study the intestinal-brain transmission mechanism of Parkinson's disease, the association between intestinal flora and Parkinson's disease, or to screen anti-Parkinson's disease drugs.

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